My research activities and efforts during my undergraduate, post-baccalaureate, graduate, and postdoctoral training are strong indications of my commitment to a career in neuroscience research. My immediate goal is to acquire sufficient support that will facilitate my endeavors to carry out the studies outlined in this K22 Career Development Award and develop the necessary skills to successfully establish an independent research program. To reach my long term goal of establishing and directing my own research laboratory, I have assembled a team of mentors who are committed to my career development and research success. I have worked closely with the primary mentor, Dr. Tatyana Svitkina, to design a Career Development Plan (CDP) that complements my prior training and will aid me in the successful pursuit of my long-term career goal. Specifically, the CDP involves training activities designed to: (1) Enhance critical thinking and creative problem solving skills; (2) Promote effective paper and grant writing skills; (3) Hone oral presentation skills and promote collegial and collaborative interactions; (4) Promote strategic planning and laboratory management skills; (5) Teach Scientific Independence; (6) Enhance mentoring and teaching skills; (7) Teach appropriate biological principles and techniques to explore the molecular and cytoskeletal mechanisms for building specialized membrane domains in neurons. To ensure that the CDP serves its intended purpose, the Mentoring Team/Advisory Committee will meet with me on a monthly and annual basis to discuss my progress toward completing the proposed research objectives outlined in the Research Strategy Section of the proposal and demonstrating the values and skills outlined in the CDP. ENVIRONMENT. The University of Pennsylvania (Penn) and the Department of Biology in Penn's School of Arts and Sciences, where I am currently being trained as a postdoctoral researcher, are strongly dedicated to research, education, and training of young scientists. The Department, as well as other schools and centers at Penn, organizes various career-promoting activities, such as professional-training workshops, research seminars, journal clubs, and student/postdoctoral training in teaching. Also, the geographical proximity of the Department to other schools and centers at Penn facilitates a collaborative environment and allows me access to a host of research facilities that will afford me the tools and resources required to successfully execute the research proposed in this application. RESEARCH. There is a fundamental gap in understanding how the axon initial segment (AIS) regulates action potential initiation and maintains neuron polarity, partly due to poor understanding of its molecular architecture and contribution of key cytoskeletal components, ankyrinG, ?IV-spectrin, and actin filaments. Continued existence of this gap represents an important problem because, until it is filled, understanding neurological disorders that occur as a result of AIS disruption through neuronal injury or mutation of AIS proteins will largely remain incomprehensible. The objective of this application is to determine the relative arrangement of fundamental AIS components that form the core framework of the AIS cytoskeleton and define specific roles for ?IV-spectrin. The central hypothesis is that ?IV-spectrin, ankyrinG and actin filaments form an extensive, interconnected network within the AIS coat that contributes to AIS membrane properties and/or function. The rationale of my proposed research is that once it is known how individual AIS components contributes to AIS cytoskeletal structure and function, it will be better understood how the AIS, and possibly neuron polarity, might be preserved or restored in the context of neuronal injury or disease. In order to test the central hypothesis and accomplish the objective of this proposal, the following specific aims will be pursued: 1) Determine the spatial arrangement of key AIS coat cytoskeletal components, ?IV-spectrin, ankyrinG, and actin filaments (current research, Phase I); and 2) Determine the role of ?IV-spectrin in AIS cytoskeletal structure and function (Phase II). This hypothesis will be tested by genetic manipulation techniques (RNAi, mutant genes, and dominant-negative constructs) to remove AIS proteins that are not part of the core ankyrinG, ?IV- spectrin, and actin filament cytoskeleton. This will generate a simplified version of the AIS that will facilitate detailed structural analysis using platinum replica electron microscopy (PREM), immunoPREM, and electron tomography. The research in the proposal is innovative because it employs a combinatory approach that represents a new way for resolving the basic AIS cytoskeletal architecture, addresses new hypotheses, and, therefore, allows acquisition of new knowledge. This proposed research is significant because it is expected to vertically advance and expand understanding on how disruption of the molecular organization of the AIS leads to different neurological conditions. Once such knowledge is available it is expected to promote our understanding of how the AIS can be restored or preserved following nervous system injury, which will preserve a neuron's polarity and ability to generate action potentials. Furthermore, better fundamental understanding of how the membrane cytoskeleton is organized in other ankyrin-spectrin based membrane domains (e.g., nodes of Ranvier, unmyelinated axons, cardio myocyte T-tubules and intercalated disks, epithelial lateral membranes, costumers, and photoreceptor inner/outer segments) can be anticipated.